Immuno stimulant substances derived from bovine casein and compositions containing the same

ABSTRACT

New immunostimulant substances are obtained by hydrolysing delipidized bovine casein with a proteolytic enzyme and fractionating the product.

The present invention relates to biologically active substances obtained by fractionation of enzymatic hydrolysates of bovine casein, and the compositions which contain them.

The present invention provides novel immunostimulant substances obtained by partial enzymatic hydrolysis of delipidized bovine casein, fractionation of the hydrolysis products according to their average molecular weights, and separation of a fraction or fractions having immunostimulant properties.

The new substances are immunological agents which, in particular, promote antibody production. The substances which have an average molecular weight between 300 and 2,500 possess especially useful properties.

After delipidization the bovine casein is subjected to the action of a proteolytic enzyme, preferably trypsin, chymotrypsin or another similar enzyme, or both trypsin and chymotrypsin. The hydrolysis products are then fractionated. For example, fractionation of the water-soluble fraction obtained by delipidizing bovine casein with a chloroform/methanol mixture and then digesting the casein with non-pretreated trypsin, yields three biologically active fractions hereinafter called IV, V and VI.

After purification on an ion-exchange column followed by high-performance liquid chromatography under specified conditions, the combined fractions "IV+V" yield the substances referred to herein as "MJH 320", "MJH 324", "MJH 328" and "MJH 329", the immunological properties of which are improved relative to those of the fractions IV and V themselves.

Subsequent purification of the substance "MJH 320" makes it possible to isolate, for example, the pentapeptide Phe-Phe-Ser-Asp-Lys, which corresponds to peptide 17-21 of bovine para-kappa-casein [J. Jolles et al., Chimia 26 (12) 645-646 (1972)].

Subsequent purification of the substance "MJH 328", or a more widely spread fractionation of the fraction which gives rise to the substance "MJH 328", makes it possible to separate, for example, the fractions "MJH 362" and "MJH 365", from which there can be respectively isolated tripeptide Leu-Leu-Tyr which corresponds to peptide 191-193 of bovine β-casein [R. Ribadeau-Dumas et al., Eur., J. Biochem., Eur., 25, 505-514 (1972)] and the hexapeptide Thr-Thr-Met-Pro-Leu-Trp which corresponds to the C-terminal peptide of bovine αS₁ -casein [J. C. Mercier et al., Eur. J. Biochem., 23, 41-51 (1971)].

The accompanying drawings illustrate the fractionation of the hydrolysed casein using the techniques described in the Examples below.

FIG. 1 is a filtration diagram illustrating the separation as described in Example 1 below, of the biologically active fractions designated MJH 274, MJH 275 and MJH 276.

FIG. 2 is a filtration diagram illustrating the separation as described in Example 1 below, of the biologically active fractions designated MJH 310, MJH 311, MJH 312, MJH 313 and MJH 314.

FIG. 3 is a filtration diagram illustrating the separation, as described in Example 1 below, of the active fractions designated MJH 328 and MJH 329.

FIG. 4 is an elution diagram illustrating the separation of the biologically active fractions MJH 320 and MJH 324 as described in Example 1 below.

FIG. 5 is an elution diagram illustrating the separation, as described in Example 3 below, of the fractions designated MJH 362 and MJH 365.

FIG. 6 is an elution diagram showing the separation, as described in Example 3 below, of the hexapeptide Thr-Thr-Met-Pro-Leu-Trp.

FIG. 7 is an elution diagram showing the separation, as described in Example 4 below, of the active fractions MJH 143 which contains the tripeptide Leu-Leu-Tyr, MJH 414 and MJH 417.

The new substances of the present invention are immunological agents which promote antibody production and accelerate the phenomenon of phagocytosis.

In vitro, they have proved especially active at concentrations between 0.1 and 10 μg/ml in the test of anti-sheep red cell (haemolytic) antibody secretion by mouse spleen cells immunised in vivo, and in the test of phagocytosis of opsonised sheep red cells by mouse peritoneal macrophages.

The Examples which follow show how the invention can be put into practice.

EXAMPLE 1

Bovine casein (500 mg) is delipidised by treatment with a chloroform/methanol (2:1 by volume) mixture (25 cc).

The casein is dissolved in 0.05M sodium hydroxide solution so that the final casein concentration is about 2 mg/cc.

The solution obtained is subjected to dialysis against 0.033M phosphate buffer (2 liters) at pH 8 for 2 days, renewing the buffer solution 5 times.

There is thus obtained a solution at pH 8 containing soluble casein. This solution is subjected to the action of trypsin at an enzyme/substrate ratio in the region of 1:100. The enzymatic hydrolysis is continued for 24 hours at 37° C., half the amount of enzyme being introduced at the beginning of the reaction and the remainder 4 hours after the start of digestion.

The reaction mixture is brought to dryness and then taken up in 30% strength acetic acid solution (16 cc). The mixture is subjected to centrifugation at 15,000 rpm for 45 minutes.

The clear supernatant liquid (16 cc) is filtered on a Sephadex G 50 column (height 117 cm, diameter 4.5 cm), eluting with 30% strength acetic acid and collecting 3 cc fractions.

Working in this manner and following the chromatography by UV absorption at 280 nm, there are obtained zones which enable 6 fractions to be defined for which the average molecular weight is established The filtration diagram is shown in FIG. 1 of the accompanying drawings.

The biologically active fractions are as follows:

substance IV (MJH 274): fractions 1,248 cc to 1,419 cc; average molecular weight 1,000±250

substance V (MJH 275): fractions 1,420 cc to 1,554 cc; average molecular weight 600±250

substance VI (MJH 276): fractions 1,555 cc to 1,695 cc; average molecular weight 400±1,000.

The combined substances IV and V (1.5 g originating from 3 successive operations) are filtered on a CM Trisacryl (IBF registered trademark) column (height 110 cm, diameter 2.2 cm), eluting with a 0.01M Tris-HCl [tris(hydroxymethyl)aminomethane, HCl] buffer solution at pH 4.5 (1,090) cc) and then with a 0.01M Tris-HCl buffer pH 4.5, 0.1M NaCl solution (1,040 cc), the fractions collected being 3.35 cc.

The filtration diagram is shown in FIG. 2, in which the elution volume appears on the abscissae and the optical densities as ordinates. 11 fractions are collected, among which the fractions eluted at 335 to 435 cc (MJH 310), 435 cc to 570 cc (MJH 311), 570 cc to 787 cc (MJH 312), 787 cc to 1,010 cc (MJH 313) and 1,306 cc to 1,374 cc (MJH 314) proved to be active.

The fractions MJH 311 and MJH 313 were purified successively by reversed phase HPLC on a semi-preparative column (Waters C 18-μ-bondapak column) the length of which is 30 cm and the diameter 7.8 mm. 1 cc fractions are collected, the elution rate being 1 cc/minute. At the start, the column is buffered with 0.1% strength trifluoroacetic acid (TFA) (eluant A). An eluant containing TFA (0.1% by volume) and acetonitrile (70%) (eluant B) is prepared. The fraction MJH 311 is dissolved in 0.1% strength TFA (1 cc).

The elution is performed using a linear elution gradient, proceeding according to the following table:

    ______________________________________                                         Time                                                                           (minutes)      Eluant A Eluant B                                               ______________________________________                                          25            100       0                                                     115             0       100                                                    ______________________________________                                    

The elution diagram is shown in FIG. 3. The elution is followed by measuring the absorption at 280 nm.

The elution diagram includes 7 zones, in which the most important fractions are the fractions eluted at 74 to 79 min (MJH 328) and 79 to 86 min (MJH 329).

Fraction MJH 313 is dissolved in 0.1% strength TFA (1 cc). The elution is performed using the elution gradient described in the following table:

    ______________________________________                                         Time                                                                           (minutes)                                                                               Eluant A      Eluant B Gradient                                       ______________________________________                                          25      100            0       isocratic                                       55      65            35       linear                                         115      65            35       isocratic                                      145       0            100      linear                                         ______________________________________                                    

The elution diagram is shown in FIG. 4. The elution is followed by measuring the absoprtion at 280 nm.

The elution diagram includes 12 zones. Active substances were demonstrated in the zones eluted at 67 to 69 min (MJH 320) and 89 to 98 min (MJH 324).

EXAMPLE 2

The substance "MJH 320" is purified by reversed phase HPLC on a semi-preparative column (Waters C 18-μ-bondapak column), the length of which is 30 cm and the diameter 7.8 mm. 0.5 cc fractions are collected, the elution rate being 1 cc/minute. At the start, the column is buffered with 0.1% strength trifluoroacetic acid (TFA) (eluant A). An eluant containing TFA (0.1% by volume) and acetonitrile (70%) (eluant B) is prepared. The fraction MJH 320 is dissolved in 0.1% strength TFA (1 cc).

The elution is performed using a step-wise isocratic elution gradient, proceeding according to the following table:

    ______________________________________                                         Time                                                                           (minutes)      Eluant A Eluant B                                               ______________________________________                                          0             100       0                                                     20             85       15                                                     60             83       17                                                     100            78       22                                                     ______________________________________                                    

The elution is followed by measuring the absorption at 220 nm.

The fractions eluted at between 29 and 42 minutes are combined and purified again under the same conditions, using the elution gradient described in the following table:

    ______________________________________                                         Time                                                                           (minutes)                                                                               Eluant A      Eluant B Gradient                                       ______________________________________                                          0       100            0                                                      20       84            16       linear                                         80       84            16       isocratic                                      110      83            17       isocratic                                      ______________________________________                                    

The elution is followed by measuring the absorption at 220 nm.

The fractions eluted at 34 to 43 cc are combined and contain the pentapeptide Phe-Phe-Ser-Asp-Lys.

The structure of the pentapeptide is determined:

by complete hydrolysis with 6N hydrochloric acid at 110° C. for 18 hours, which shows the presence of phenylalanine (Phe)=2, serine (Ser)=1, aspartic acid (Asp)=1 and lysine (Lys)=1

by dansylation, so as to determine the nature of the N-terminal amino acid, which is phenylalanine

by enzymatic digestion with carboxypeptidase B, so as to determine the nature of the C-terminal amino acid which is lysine

by analysis with the Beckmann Model 890 C sequenater, 0.1M Quadrol program, with determination of the different stages by characterisation of the amino acid phenylthiohydantoin derivatives (determination by HPLC and visualisation on plates).

EXAMPLE 3

The fraction "MJH 311" obtained in Example 1 is purified by reversed phase HLPC on a semi-preparative column (Waters C 18-μ-bondapak column), the length of which is 30 cm and the diameter 7.8 mm. 1 cc fractions are collected, the elution rate being 1 cc/minute. At the start, the column is buffered with 0.1% strength trifluoroacetic acid (TFA) (eluant A). An eluant containing TFA (0.1% by volume) and acetonitrile (70%) (eluant B) is prepared. The fraction "MJH 311" is dissolved in 0.1% strength TFA (1 cc).

The elution is performed using an elution gradient, proceeding according to the following table:

    ______________________________________                                         Time                                                                           (minutes)                                                                               Eluant A      Eluant B Gradient                                       ______________________________________                                         15       100            0       isocratic                                      40       70            30       linear                                         75       65            35       linear                                         105      65            35       isocratic                                      ______________________________________                                    

The elution diagram is shown in FIG. 5. The elution is followed by measuring the absorption at 280 nm.

The elution diagram includes 4 zones, the most important fractions of which are the fractions eluted at 61 to 63 minutes (MJH 362) and 69 to 73 minutes (MJH 365). The fraction "MJH 365" corresponds to the main peak of the fraction "MJH 328" shown in FIG. 3.

The fraction "MJH 365" is purified by HPLC using a semi-preparative column described above with the same eluants and at the same flow rate, collecting 0.5 cc fractions. The fraction "MJH 365" is dissolved in 0.1% strength TFA (1 cc).

The elution is performed using an elution gradient, proceeding according to the following table:

    ______________________________________                                         Time                                                                           (minutes)                                                                               Eluant A      Eluant B Gradient                                       ______________________________________                                          0       100            0                                                      30       75            25       linear                                         50       75            25       isocratic                                      100      73            27       isocratic                                      ______________________________________                                    

The elution diagram is shown in FIG. 6. The elution is followed by measuring the absorption at 280 nm.

The fractions eluted between 74.5 and 79 minutes are combined and contain the hexapeptide Thr-Thr-Met-Pro-Leu-Trp.

The structure of the hexapeptide is determined:

by complete hydrolysis with 6N hydrochloric acid at 110° C. for 18 hours, which shows the presence of threonine (Thr)=2, methionine (Met)=1, proline (Pro)=1, leucine (Leu)=1

by hydrolysis with 4N methanesulphuric acid containing 0.2% of tryptamine at 100° C. for 18 hours under reduced pressure which shows the presence of tryptophan (Trp)=1

by dansylation so as to determine the nature of the N-terminal amino acid, which is threonine

by enzymatic digestion with the carboxypeptidase A so as to determine the nature of the C-terminal amino acid, which is tryptophan.

by analysis with the Beckmann Model 890 C sequenator, 0.1M Quadrol program.

EXAMPLE 4

The fraction "MJH 362" separated in Example 3 is purified by HPLC under the conditions described above, collecting 0.5 cc fractions.

The elution is performed using an isocratic gradient, proceeding according to the following table:

    ______________________________________                                         Time                                                                           (minutes)      Eluant A Eluant B                                               ______________________________________                                          0             100      0                                                      15             100      0                                                      75              82      18                                                     ______________________________________                                    

The elution diagram is shown in FIG. 7. The elution is followed by measuring the absorption at 280 nm.

The elution diagram includes 6 zones, the most important of which are eluted at 38.5 to 43 cc (MJH 413), 43.5 to 47 cc (MJH 414) and 52 to 53.5 cc (MJH 417).

The fraction MJH 413 contains the tripeptide Leu-Leu-Tyr. The structure of the tripeptide is determined:

by complete hydrolysis with 6N hydrochloric acid at 110° C. for 18 hours which shows the presence of leucine (Leu)=2 and tyrosine (Tyr)=1

by dansylation so as to determine the nature of the N-terminal amino acid which is leucine

by the action of carboxypeptidase A so as to determine the nature of the C-terminal amino acid, which is tyrosine.

The present invention also provides pharmaceutical compositions which can be used in therapy containing a substance according to the present invention in combination with one or more diluents or adjuvants which are compatible and pharmaceutically acceptable.

These compositions can be used as vaccine adjuvants (for example for anti-influenza vaccine composed of haemagglutinating sub-units, anti-poliomyelitis vaccine with inactivated virus, antimalarial vaccine), injected simultaneously with the antigen (viral, bacterial, parasitic, fungal, tumour) in respect of which it is desired to increase antibody production or specific cell reactivity.

These pharmaceutical compositions can also be used as non-specific immunostimulants, with a view to increasing the resistance of the host (man or domestic animal) to infections or in anti-tumour immunotherapy.

As adjuvants, the new substances can be administered either in aqueous solution, or in oily emulsion, or alternatively in the form of liposomes with the antigen in respect of which it is desired to obtain an increased or improved immune response, by the route used for this antigen and in proportions which vary between 0.01 and 10 times the amount of antigen with which they are mixed before being injected.

For application as a non-specific immunostimulant, the new substances can be used intravenously, intramuscularly, subcutaneously, intranasally, and optionally orally or rectally, either in aqueous solution or in oily emulsion, or alternatively in the form of liposomes. In this case, the dose of substance according to the invention which is administered is generally between 0.01 and 10 mg/kg. In human therapy, the daily dosage depends on the effect sought. It can be between 0.5 and 10 mg for an adult.

Solid compositions for oral administration can be tablets, pills, powders or granules.

Liquid compositions for oral administration can be pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.

The compositions for parenteral or intranasal administration can be sterile aqueous solutions, or suspensions or emulsions.

Sterilisation can be carried out in several ways, e.g. by means of a bacteriological filter or by incorporating sterilising agents. The solid compositions made sterile by irradiator (β-rays) can be dissolved in sterile water or any other injectable sterile medium, optionally at the time of use.

The compositions for rectal administration are suppositories.

The Example which follows illustrates a composition according to the invention.

EXAMPLE

By customary technique, a liquid composition which can be administered intravenously is prepared, having the following composition:

    ______________________________________                                         substance MJH-320      10    mg                                                injectable solution    5     cc                                                ______________________________________                                     

We claim:
 1. An immuno stimulant substance having an average molecular weight in the range 300 to 2,500 and obtained by partial enzymatic hydrolysis of delipidized bovine casein, chromatographic fractionation of the hydrolysis products according to their average molecular weights, and separation of a fraction or fraction having immuno stimulant properties wherein the proteolytic enzyme used is trypsin, chymotrypsin or both trypsin and chymotrypsin.
 2. An immunostimulant substance according to claim 1 wherein the casein is hydrolysed for about 24 hours at 37° C. using an enzyme/substrate ratio of about 1:100.
 3. An immunostimulant substance according to claim 1 wherein the casein is delipidized by treatment with a mixture of chloroform and methanol.
 4. An immunostimulant substance according to claim 1 wherein the fractionation of the hydrolysis products is effected by gel filtration.
 5. An immunostimulant substance according to claim 4 wherein the fractionation is followed by ion-exchange chromatography and high performance liquid chromatography.
 6. A pharmaceutical composition comprising an effective amount of an immunostimulant substance according to claim 1 in association with a compatible, pharmaceutically acceptable diluent or adjuvant. 